冷冻干燥法制备细胞微载体研究

发布时间：2018-07-28 16:50

【摘要】：肝功能衰竭是临床上极具挑战性的一种病症，虽然肝移植可挽救许多肝病末期的患者的生命，但是由于肝脏供体的短缺等原因，许多患者在等待肝移植的过程中死亡。20世纪逐渐发展的生物人工肝支持系统，主要是用来为肝衰竭患者提供体外肝脏功能支持的方法，经过多年的发展，生物人工肝技术已经逐渐成熟，成为肝衰竭患者治疗的新方法之一。本课题旨在研究生物人工肝中肝细胞培养所需的微载体的制备工艺，制备出性能良好的多孔微载体，并培养肝细胞，验证其良好的生物相容性，为临床上肝病的治疗打下基础。 1.以壳聚糖为基质，通过共混法引入海藻酸钠或N-羧丙酰壳聚糖钠，采用乳液冷冻干燥法，并结合正戊醇和醋酸铵作致孔剂来制备海藻酸钠/壳聚糖多孔微载体和N-羧丙酰壳聚糖钠/壳聚糖微载体，，在该微载体上培养人肝细胞L-02。 2.用扫描电子显微镜观察微载体的微观结构形貌，以吸水率、体外降解率、MTT比色等指标，综合评价海藻酸钠/壳聚糖微载体和N-羧丙酰壳聚糖钠/壳聚糖微载体的性能及生物活性。 3.对于N-羧丙酰壳聚糖钠/壳聚糖微载体，选择的致孔剂不同，所得的微载体的表观形貌也不同。以正戊醇为致孔剂，冻干得到的微载体孔径为3-55m，孔隙率为88%；而以正戊醇和醋酸铵两种致孔剂得到的支架孔径为15-55m，孔隙率为94%。两种方法得到的支架硬度高，溶胀性良好，吸水率高，两种空白N-羧丙酰壳聚糖钠/壳聚糖支架在体外可完全降解。光学显微镜观察L-02人肝细胞在N-羧丙酰壳聚糖钠/壳聚糖支架上生长良好。 4.对于海藻酸钠/壳聚糖微载体，以正戊醇为致孔剂，冻干得到的微载体孔径为3-50m，孔隙率为89%；以正戊醇和醋酸铵两种致孔剂制孔得到的微载体孔径为15-55m，孔隙率为95%。且两种方法得到的微载体硬度高，溶胀性良好，吸水率高，空白海藻酸钠/壳聚糖微载体在体外可完全降解。光学显微镜观察L-02人肝细胞在海藻酸钠/壳聚糖微载体上生长良好。综上所述，本课题所制备的多孔微载体具有良好的生物学性能，可满足生物人工肝对肝细胞培养的要求。
[Abstract]:Liver failure is a very challenging disease in clinic. Although liver transplantation can save the lives of many patients at the end of liver disease, it is due to the shortage of liver donors and so on. Many patients died while waiting for liver transplantation. The biological artificial liver support system developed gradually in the 20th century, mainly used to provide in vitro liver function support for patients with liver failure, after years of development. Biological artificial liver technology has gradually matured and become one of the new methods for the treatment of liver failure patients. The purpose of this paper is to study the preparation of microcarriers for hepatocyte culture in bioartificial liver, to prepare porous microcarriers with good performance, and to culture liver cells to verify their good biocompatibility. For the clinical treatment of liver disease lay the foundation. 1. Sodium alginate or sodium N-carboxypropionyl chitosan was introduced by blending with chitosan as the matrix, and the emulsion freeze-drying method was used. Sodium alginate / chitosan porous microcarriers and N-carboxypropionyl chitosan / chitosan microcarriers were prepared by using n-pentanol and ammonium acetate as pore-inducing agents. L-02.2 human hepatocytes were cultured on the microcarriers. The microstructure and morphology of microcarriers were observed by scanning electron microscope (SEM). The indexes of water absorption, in vitro degradation rate and MTT colorimetric analysis were used. The properties and biological activities of sodium alginate / chitosan microcarriers and N-carboxypropionyl chitosan / chitosan microcarriers were comprehensively evaluated. For N-carboxypropionyl chitosan / chitosan microcarriers, the morphology of the microcarriers was different with different pore-forming agents. Using n-pentanol as the pore-inducing agent, the pore size of the microcarrier was 3-55m and the porosity was 88m, while the pore size of the scaffold was 15-55m and the porosity of the scaffold was 9455 m. The scaffolds obtained by the two methods have high hardness, good swelling and high water absorption. The two blank N-carboxypropionyl chitosan / chitosan scaffolds can be completely degraded in vitro. L-02 human hepatocytes grew well on N-carboxypropionyl chitosan / chitosan scaffolds under optical microscope. For sodium alginate / chitosan microcarrier, the pore size of freeze-dried microcarrier was 3-50 m, the porosity was 89 m, and the pore size of microcarrier was 15-55 m with 95% porosity by using n-pentanol and ammonium acetate as pore-forming agent. The microcarriers obtained by the two methods have high hardness, good swelling and high water absorption. The blank sodium alginate / chitosan microcarriers can be completely degraded in vitro. L-02 human hepatocytes grew well on sodium alginate / chitosan microcarriers under optical microscope. In conclusion, the porous microcarriers prepared in this paper have good biological properties and can meet the requirements of bioartificial liver for hepatocyte culture.
【学位授予单位】：上海理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

【参考文献】